localized treatment with oncolytic adenovirus delta-24 ......2019/08/27 · 1 localized treatment...

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Localized Treatment with Oncolytic Adenovirus Delta-24-RGDOX Induces Systemic Immunity against

Disseminated Subcutaneous and Intracranial Melanomas

Hong Jiang1, Dong Ho Shin

1, Teresa T. Nguyen

1, Juan Fueyo

1, Xuejun Fan

1, Verlene Henry

1, Caroline C.

Carrillo2, Yanhua Yi

1, Marta M. Alonso

3, Tiara L. Collier

1, Ying Yuan

4, Frederick F. Lang

1, and Candelaria

Gomez-Manzano1

1Brain Tumor Center;

2Applied Cancer Science Institute;

4Department of Biostatistics; The University of Texas

MD Anderson Cancer Center, Houston, Texas. 3University Hospital of Navarra, Pamplona, Spain.

Running Title: Delta-24-RGDOX induces systemic anti-melanoma immunity

Keywords: oncolytic adenovirus, Delta-24-RGDOX, immunity, melanoma, metastasis

Financial Support: This work was supported in part by NIH/NCI grants P50CA127001 and Cancer Center

Support Grant P30CA016672 (Research Animal Support Facility, Small Animal Imaging Facility); the Cancer

Prevention and Research Institute of Texas (RP170066); the Department of Defense (Team Science Award

CA160525); the Marnie Rose Foundation; the J.P. Harris Brain Tumor Research Fund; the Bradley Zankel

Foundation; the Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (Parent

F31: 1 F31 CA228207 01A1), the American Legion Auxiliary Fellowship in Cancer Research; Terry and Janet Klebe

Fellowship.

Corresponding Authors:

Hong Jiang, Department of Neuro-Oncology, Unit 1002, The University of Texas MD Anderson Cancer Center,

1515 Holcombe Blvd., Houston, TX 77030. Phone: 713-834-6203; Email: [email protected].

Research. on June 17, 2021. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 27, 2019; DOI: 10.1158/1078-0432.CCR-19-0405

mailto:[email protected]://clincancerres.aacrjournals.org/

2

Candelaria Gomez-Manzano, Department of Neuro-Oncology, Unit 1002, The University of Texas MD

Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: 713-834-6221; Email:

[email protected].

Disclosure of Potential Conflicts of Interest: HJ, FFL, JF, and CGM are reporting ownership interest

(including patents). CGM and JF are consultants and shareholders of DNATrix, Inc. . The authors have no

other financial interests.

Word Count: 4997

Number of Figures: 6



mailto:[email protected]://clincancerres.aacrjournals.org/

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Translational Relevance

Immune checkpoint blockade revolutionized the therapy for melanoma. But the therapeutic benefit is

limited to only a subset of patients with immunogenic (“hot”) tumors and is restrained by immune-related

adverse events. We tested the effect of injecting oncolytic adenovirus Delta-24-RGDOX into primary

subcutaneous melanoma on disseminated subcutaneous and intracranial melanomas in syngeneic mouse models.

We observed systemic immune activation in the treated mice, resulting in the regression of both treated and

untreated tumors. Thus, the virus is expected to be efficacious in patients with immunosuppressive (“cold”)

tumors as well and, given its cancer-selective property and localized application, have better safety profile in

cancer patients than immune checkpoint blockade. Importantly, this is the first report to demonstrate that

localized treatment with an oncolytic virus in the subcutaneous tumor is able to reject intracranial tumors,

suggesting Delta-24-RGDOX can be applied to patients with brain melanoma metastasis.



http://clincancerres.aacrjournals.org/

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Abstract

Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious anti-

glioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is

effective against disseminated melanomas.

Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary

subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6

mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored

with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry.

Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited

the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging

survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment

increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated

the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor.

Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-

injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells

and their migration to tumors expressing the target antigen.

Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination

of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in

systemic immunity against disseminated s.c. and i.c. tumors.




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Introduction

Because of its propensity to metastasize throughout the body, melanoma is a deadly tumor, accounting

for the majority (60–80%) of deaths from skin cancer (1, 2). Ninety-five percent of melanoma patients with 3 or

more sites of metastatic disease die within 1 year (3). Over the past decade, immune checkpoint blockade (ICB)

has significantly improved the clinical outcomes of melanoma patients. However, the clinical benefit of ICB is

preferentially achieved in only a subset of patients with a pre-existing T-cell response against the tumor (“hot”

tumor) (4-8). Moreover, because checkpoint receptors play important regulatory roles in autoimmunity, the

toxicity of ICB therapies causes immune-related adverse events (IRAEs). The IRAE incidence ranges from 70%

in patients treated with αPD-1/αPD-L1 antibodies to as high as 90% in patients treated with αCTLA-4 (9).

Therefore, safe and efficacious therapies are imperatively needed.

Oncolytic viruses are a new class of immunotherapy drugs (10). They are genetically modified or

naturally occurring viruses that selectively replicate in and disrupt cancer cells (11-13). In 2015, talimogene

laherparepvec (T-VEC), a modified herpes simplex virus type 1 with cancer-selective replication and

granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, became the first oncolytic virus

approved by U.S. Food and Drug Administration (FDA) for the treatment of surgically unresectable skin and

lymph node lesions in patients with advanced melanoma (14, 15). In a randomized open-label phase III trial,

those in the T-VEC arm achieved a better durable response rate than those in the control arm did (16% versus

2%; median overall survival duration, 23.3 versus 18.9 months) (14). Thus, even though T-VEC has not

dramatically improved melanoma therapy, it marks the beginning of a new era in which metastatic cancers can

be treated with localized intratumoral injections of oncolytic viruses, which induced the regression of both the

virus-injected and distant tumors, including visceral metastases (16).

In a recent phase I clinical trial based on our long-term effort to develop oncolytic adenoviruses for

cancer therapy, an intratumoral injection of Delta-24-RGD (DNX-2401, Tasadenoturev), a second-generation

oncolytic adenovirus that is derived from human adenovirus serum type 5 and targets tumor cells with an

aberrant RB pathway (17-19), elicited inflammation within the injected tumors, leading to a durable complete




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response in 12% of patients with recurrent glioblastoma (20). To further increase the efficacy of the virus, we

developed a next-generation oncolytic adenovirus, Delta-24-RGDOX, which expresses the immune stimulatory

OX40 ligand (OX40L). Compared with its predecessor, this new virus elicits a more potent autologous cancer

vaccination in situ, resulting in an efficacious, tumor-specific, long-lasting therapeutic effect in

immunocompetent mouse glioma models (21). Because the virus preferentially expresses OX40L on the surface

of infected tumor cells, thereby stimulating activated T cells that recognize the antigens presented on the tumor

cells (21), Delta-24-RGDOX may be more cancer-specific and have a better safety profile in patients than T-

VEC, which secrets GM-CSF in the tumor milieu (16). A clinical trial of Delta-24-RGDOX in glioma patients

has been approved in Spain and is pending approval by the U.S. FDA.

In our experience, Delta-24-RGD and Delta-24-RGDOX induce T-cell infiltration in the virus-injected

glioma and immune memory against the treated tumor (20-22), suggesting that the viruses are able to turn

immune-suppressive (“cold”) tumors into immune-active (“hot”) tumors. We hypothesized that Delta-24-

RGDOX has the same effect in melanoma and that the virus-mediated therapeutic effect could extend to

untreated disseminated/metastatic tumors. To test this hypothesis, we examined the virus in subcutaneous

(s.c.)/s.c. and s.c./i.c. syngeneic melanoma models in immunocompetent mice. We found that injections of

Delta-24-RGDOX not only induced immunity against the virus-injected s.c. tumor but also affected distant

untreated tumors and changed the immune landscape in the treated mice. For the first time, we demonstrated

this type of virotherapy induced efficacious immunity against melanoma in brain. Our findings suggest that

localized treatment with Delta-24-RGDOX can be an effective therapeutic agent for patients with metastatic

tumors, including brain metastasis.

Materials and Methods

Cell lines and culture conditions

Human lung carcinoma A549 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium

nutrient mixture F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories,




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Inc.), 100 μg/ml penicillin, and 100 μg/ml streptomycin. The mouse melanoma cell lines B16-F10 (ATCC),

B16F10 Red-FLuc (PerkinElmer), B16F10 Red-FLuc-3 (a subclone of B16F10 Red-FLuc with slower growing

s.c. and i.c. tumors), and B16-ovalbumin (OVA; a kind gift from Dr. Hong Qin, MD Anderson Cancer Center)

(23, 24) were grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics. Human embryonic

kidney 293 cells (QBioGene, Inc.) human melanoma A375 cells (ATCC) and mouse lung carcinoma CMT64

cells (Culture Collections, Public Health England, UK) were maintained in DMEM supplemented with 10%

FBS and antibiotics. All cells were kept at 37°C in a humidified atmosphere containing 5% CO2. Experiments

were carried out within 6 months after the cell lines were obtained from a cell bank or within 15 passages in the

laboratory. All cell lines were tested for and found to be free of mycoplasma.

B16-F10enhanced green fluorescent protein (EGFP) cells

First, EGFP cDNA (Clontech) was subcloned into the lentivector plasmid pCDH-CMV-MCS-EF2-Puro

(System Biosciences). The plasmid was transduced into B16-F10 cells with X-tremeGENE HP DNA

Transfection Reagent (Millipore Sigma), and a stable cell line was established by selection with 1 µg/ml

puromycin.

Viruses

The Delta-24-RGD and Delta-24-RGDOX viruses were propagated in A549 cells and titrated in 293

cells as described previously (21).

Mice

C57BL/6 mice were provided by MD Anderson’s Mouse Resource Facility. OT-I/Luc mice (a kind gift

from Dr. J.J. Moon, University of Michigan) were bred at MD Anderson’s animal facility as described

previously (25).




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Animal studies

Six- to ten-week-old mice were used. B16-F10 cells and their derivatives (5 × 105

cells/mouse) were

grafted subcutaneously onto the backs of the mice. B16F10 Red-Fluc-3 (2,000 cell/mouse) were grafted into the

caudate nucleus of the mice using a guide-screw system as described previously (18). One day before virus

treatment, the mice with implanted tumors were evaluated with bioluminescent imaging to assess tumor growth.

Mice with off-size tumors were eliminated from the study, and the rest of the mice were randomly assigned to

experimental groups. Delta-24-RGDOX (2 × 108

plaque-forming units [PFUs]/mouse) was injected into the first

implanted s.c. tumor. For rechallenging the surviving mice, B16F10 Red-FLuc (5 × 105

cells /mouse) or CMT64

(1× 106

cells /mouse) cells were implanted s.c.; or B16F10 Red-FLuc-3 (2,000

cells /mouse) cells were

implanted in the contralateral hemisphere of the mouse brain. For monitoring tumor-specific T cells in vivo, 1×

106

OT-I/Luc CD8+ T cells /mouse were injected into the primary s.c. tumor one day before the virotherapy. In

the s.c./s.c. model, mice were euthanized when the sum of the longer diameters of the two s.c. tumors was ≥ 2

cm. In the s.c./i.c. model, mice were euthanized when either the longer diameter of s.c. tumor was ≥ 2 cm or the

mice showed neurological disorders, such as hunched posture, loss of weight and slow movement. All animal

studies were conducted in C57BL/6 mice. All experimental procedures involving the use of mice were done in

accordance with protocols approved by MD Anderson’s Animal Care and Use Committee and followed U.S.

National Institutes of Health and Department of Agriculture guidelines.

Bioluminescence imaging of tumor-bearing mice

Following implantation of the bioluminescent cells, the luminescence from the growing tumors in the

mice was detected with the IVIS Spectrum imaging system (PerkinElmer). Briefly, each mouse was injected

subcutaneously with 200 μl D-luciferin (Gold Biotechnology; 15 mg/ml in phosphate-buffered saline [PBS]).

Ten minutes later, the animals were anesthetized by placing them in a chamber with 2% isoflurane gas in O2

until they were unresponsive. The anesthetized animals were moved to the imaging chamber to acquire images




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of the tumors. The luminescence in the mice was assessed with the Living Image software program

(PerkinElmer).

Immunofluorescence staining

Paraffin-embedded tumor tissue was cut into 5-µm sections and processed for immunofluorescence

staining with standard procedures. The primary antibodies were anti-E1A (MA1-91861; Invitrogen); the

secondary antibodies were anti-mouse IgG Alexa Fluor (A11001). Finally, the slides were mounted with cover

slips using a ProLong Gold antifade reagent (Invitrogen) containing DAPI for nuclei staining. Images were

viewed and assessed using a FluoView confocal laser scanning microscope (Olympus).

Enrichment of leukocytes from tumors

The enrichment of leukocytes from s.c. tumors was conducted as described previously with modification

(26). Briefly, the tumors were collected, placed in a 100-μm cell strainer set in petri dishes with RPMI 1640

medium, and then processed through the cell strainer into the dish. The mixture in the dish was gently pipetted

up and down and brought up to 20 ml/g of tumor. After centrifugation for 5 min at 500×g, the pellet was

resuspended in 10 ml of medium and loaded on top of a discontinuous Percoll gradient medium (44%:67%).

After centrifugation at 800×g for 30 minutes at 4°C, the cells were retrieved from the 44%-67% interphase.

Brain-infiltrating leukocytes from mouse brain hemispheres were separated from myelin debris using

Percoll gradient medium (GE Healthcare Bio-Sciences) and centrifuged as described previously (27).

Preparation of splenocytes, lymph node cells, and CD8+ T lymphocytes

Mouse spleens and lymph nodes were collected and processed as described previously, except that the

lymph node cells were not subjected to red blood cell lysis (21). CD8+ T cells were enriched from the

splenocytes with a mouse CD8a+ T Cell Isolation Kit (Miltenyi Biotec, Inc.).




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IFNγ ELISPOT assay

The ELISPOT assay was performed with Mouse IFNγ ELISpot kit from R&D systems. Briefly,

splenocytes (5 × 105 per well) were stimulated with 1 µg/ ml CD8+ T cell-specific OVA (257-264) (Invitrogen)

or GFP (118-126) peptide (Biomatik) for 21 hrs. IFNγ spot-forming cells were determined according to the

instructions from the manufacturer.

Flow cytometry analysis

To analyze cell surface protein expression, we incubated cells (2-5 × 105) in 100 µl of primary antibody

solution diluted in PBS plus 3% bovine serum albumin and 1 mM ethylenediaminetetraacetic acid (EDTA).

After incubation at 4°C in the dark for 30 minutes, the cells were washed once with 1 ml of cold PBS. Then, the

cells were resuspended in 0.5 ml of PBS. The stained cells were analyzed using flow cytometry. The antibodies

used in the studies were to the following mouse proteins: CD252 (OX40L) allophycocyanin (APC; 17-5905-

80), CD3e phycoerythrin (PE; 12-0031-81), CD4 APC (17-0041-81), CD8a PE-Cyanine5.5 (35-0081-80), PD-1

fluorescein isothiocyanate (11-9981-81), TIM3 PE-Cyanine7 (25-5870-80), CD44 super bright 600 (63-0441-

80), CD62L APC-eFlour 780 (47-0621-80), CD25 PE-Cyanine5.5 (35-0251-80), FR4 PE-Cyanine7 (25-5445-

80), MHC Class I (H-2Kb) APC (17-5958-80 ), Mouse IgG2a kappa Isotype Control APC (17-4724-81), Rat

IgG2b kappa Isotype Control (14-4031-82), MHC Class II (I-A/I-E, 14-5321-81), from eBioscience; and CD45

BV786 (564225), from BD Biosciences. The goat anti-rat IgG F(ab')2-APC (sc-3832) was from Santa Cruz

Biotechnology, Inc.. Tetramer/PE - H-2 Kb OVA (SIINFEKL) (OVA-Tet; 14087-PE Kb/OVA) was from the

MHC Tetramer Production Facility, Baylor College of Medicine. Cell counts were obtained using 123count

eBeads (01-1234, eBioscience) as a control for calculating processed sample volumes. Dead cells were

excluded from the analysis through staining with Ghost Dye Violet 510 (13-0870, Tonbo Biosciences).

Stimulation of immune cells




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For preparation of the target cells, B16F10 Red-FLuc cells were infected with the virus (100 PFU/cell).

Four hours later, 50 units/ml mouse interferon gamma (IFNγ; Prospec Protein Specialists) was added to the

culture. Forty-eight hours after viral infection, the cells were fixed with 1% paraformaldehyde and washed

before added to the co-culture. For immune cell stimulation, pre-fixed target cells (2 × 104 cells/well)

were

incubated with splenocytes or lymph node cells (5 × 105 cells/well). Forty hours after the co-culture in a round-

bottom 96-well plate, the concentration of IFNγ in the medium was assessed with a Mouse IFNγ DuoSet ELISA

kit (R&D Systems).

Statistical analysis

In quantitative studies of cultured cells and flow cytometry, each group consisted of triplicate samples.

Each study was repeated at least once. Differences between groups were evaluated using a 2-tailed Student t-

test. The animal survival curves were plotted according to the Kaplan–Meier method. Survival rates in the

different treatment groups were compared using the log-rank test. Waterfall charts were plotted with R from

http://www.R-project.org. P values < 0.05 were considered significant.

Results

Localized treatment with Delta-24-RGDOX inhibits treated primary and untreated distant s.c.

melanomas and induces immune memory

It has been reported that oncolytic adenoviruses have shown oncolytic efficacy in melanomas (28, 29).

Thus, first we tested Delta-24-RGDOX in cultured human and mouse melanoma cells. The virus infected both

human and mouse melanoma cells and expressed OX40L efficiently in these cells (Supplementary Fig. S1A).

The B16F10 and its derivatives used in our studies are comparably sensitive to Delta-24-RGDOX infection. 48

hours after viral infection at 30 pfu/cell, the percentage of OX40L positive cells ranged from 50% to 96%

among these cell lines (Supplementary Fig. S1B). Three days after the virus was injected into a s.c. melanoma

derived from B16F10-EGFP cells, OX40L expression was detected in about 11% of the tumor cells but not in




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the PBS-treated tumor (P = 0.0002; Supplementary Fig. S1C). This virus replicated in human melanoma A375

cells, albeit not as efficiently as in human lung cancer A549 cells, which are optimal hosts for adenovirus

replication. The replication of Delta-24-RGDOX was detectable in mouse melanoma B16F10 Red-FLuc cells

(Supplementary Fig. S1D) and the virus lysed the cells efficiently (Supplementary Fig. S1E).

Next, we examined the anti-melanoma activity of the virus in an s.c./s.c. syngeneic melanoma model,

derived from B16F10 Red-FLuc cells, in immunocompetent C57BL/6 mice (Fig. 1A). Monitoring of tumor

growth with bioluminescence imaging ((Fig. 1B) revealed that 3 injections of Delta-24-RGDOX into the

primary s.c. tumor inhibited both the treated tumor and untreated distant tumors (Fig. 1C, Supplementary Fig.

S2), thereby prolonging survival and resulting in a long-term survival rate of 44% in the tumor-bearing mice

(median survival durations: 36 vs 16 days, P = 0.005, Fig. 1D). The survivors of the virus treatment were

protected from rechallenging with s.c. injection of the same tumor cells but not CMT64 cells of lung carcinoma

(Fig. 1E), indicating that the treatment induced immune memory specifically against the virus-treated tumor.

Localized treatment with Delta-24-RGDOX inhibits treated s.c. primary and untreated distant i.c.

melanomas

Consistently, in the s.c./i.c. melanoma model derived from B16F10 Red-FLuc-3 cells, a subclone of

B16F10 Red-FLuc cells that grow slower than the parental cells in the i.c. tumor and give enough window

period for us to evaluate the effect of Delta-24-RGDOX in this model (Fig. 2A), viral injections into the

primary s.c. tumor inhibited not only the treated tumor but also the untreated i.c. tumor (Fig. 2B and C,

Supplementary Fig. S3), thereby prolonging survival and resulting in a long-term survival rate of 40% in the

tumor-bearing mice (median survival durations: 58 vs 27 days, P = 0.005, Fig. 2D). Since the s.c. tumor grew

faster, all the mice in PBS group were euthanized because of s.c. tumor burden and 6 of the brains showed

visible tumor. Because the virus inhibited s.c. tumor growth, in the D24-RGDOX group, of the six euthanized

mice, 4 died of brain tumor, 2 died of s.c. tumor. The survivors of the virus treatment were resistant to

rechallenging with the same type of tumor cells in the contralateral hemisphere of the brain (Fig. 2E and F),




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indicating that the treatment induced immune memory against the virus-treated tumor. Moreover, we found no

pathological changes in the brains from the survivors (Figure 2F, lower panel).

Collectively, these data demonstrate that the anti-cancer effect of intratumoral injection of Delta-24-

RGDOX could extend from the treated tumor to the distant untreated s.c. tumor and could even pass the blood-

brain barrier and reach the brain tumor, meaning that localized treatment of a primary s.c. tumor with the virus

can have a efficacious therapeutic effect on disseminated/metastatic tumors.

Intratumoral injection of Delta-24-RGDOX increases the presence of T cells and activates these cells at

the virus-injected tumor

To study the effect induced by Delta-24RGDOX within the injected tumor, we first checked the tumor

24 h after viral injection. Immunostaining revealed that the virus expressed the early protein E1A in the infected

tumor cells at this time (Fig. 3A). Two days after the viral injections, we also observed virus-mediated

upregulation of MHC I (about 6-fold increase) and MHC II (almost doubled) expression on melanoma cells

(Fig. 3B), indicating the tumor cells are more active in presenting tumor-associated antigens to the immune

cells. Three days after the viral injections, the number of leukocytes (CD45+ cells) nearly doubled in the treated

tumor (Fig. 3C). The frequency of T cells (CD45+ CD3+) tripled in the virus-treated tumors (from 20.7% to

63.4%), while the frequency of cytotoxic T cells (CD8+) increased by 23% (P < 0.0001) and that of T helper

cells (CD4+) showed little change (P = 0.9; Fig. 3D). However, owing to the increase of the leukocytes in the

tumor, the numbers of T cells, cytotoxic T cells, and helper T cells all increased dramatically in the tumors (Fig.

3E). Further analysis of the T-cell populations revealed that the virus activated T cells, thereby increasing the

frequency and number of effector (CD44+ CD62L-) CD8+ or CD4+ T cells within the virus-treated tumor (Fig.

3F). Thus, intratumoral injection of Delta-24-RGDOX caused immune activation in the tumor.

Localized treatment of the tumor with Delta-24-RGDOX causes systemic activation of T cells in tumor-

bearing mice




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Since the injection of Delta-24-RGDOX in the primary s.c. tumor mediated the regression of both the

treated tumor and disseminated untreated tumors (Fig. 1 and 2), we hypothesized that the virus could change the

entire immune landscape in the treated mice while inducing the same effect in untreated tumors. Indeed, we

found that in peripheral blood, the frequency of T cells and cytotoxic T cells increased after virus treatment (P ≤

0.001), whereas the frequency of T helper cells declined slightly (P = 0.02; Fig. 4A). The frequency of T

effector CD8+ T cells almost doubled (from 12.9% to 25.1%), whereas that of CD4+ T cells barely changed (P

= 0.08; Fig. 4B). Consistent with these findings, in the hemispheres with implanted tumors, Delta-24-RGDOX

induced the following changes: the frequency of T cells more than doubled after virus injections in the s.c.

tumor; the frequency of CD8+ T cells increased by 28% (P = 0.0001), whereas the frequency of CD4+ T cells

remained almost the same (P = 0.1; Fig. 4C); the numbers of total T cells, CD4+ T cells, and CD8+ T cells

increased by 85%137% (Supplementary Fig. S4); and the numbers of effector T cells in both the CD8+ and

CD4+ T-cell populations nearly doubled (Fig. 4D). As in peripheral blood, after virus treatment, the frequency

of effector CD8+ T cells increased 2-fold in splenocytes and increased by 60% in cells from inguinal lymph

nodes (the tumor-draining lymph nodes [TDLNs] for s.c. tumors), whereas the frequency of CD4+ T cells

increased only slightly in splenocytes and declined in TDLN cells (Supplementary Fig. S5). When splenocytes

or inguinal lymph node cells were co-cultured with melanoma cells with or without viral infection, by assessing

the IFNγ secretion into the medium, we found that the immune cells from the virus-treated group demonstrated

higher activity against the tumor cells (Fig. 4E). Furthermore, Delta-24-RGDOXinfected melanoma cells

stimulated immune cell activity more potently than Delta-24-RGDinfected cells or cells without viral infection

did (Fig. 4E), suggesting that OX40L expression on the cell surface enhances the capability of tumor cells to

induce anti-cancer immunity, which is consistent with what we observed previously in glioma virotherapy (21).

Delta-24-RGDOX disrupts immune suppression in both treated primary and untreated distant tumors

To better understand the effect of virotherapy on immunity, we further examined the status of exhausted

T cells (PD-1+ TIM3+) (30) and regulatory T cells (CD4+ CD25+ FR4+) (31). First, we analyzed these cell




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populations in the tumors. We found that Delta-24-RGDOX reduced the frequency of exhausted CD8+ T cells

in both virus-injected s.c. tumors (from 37% to 10.5%, P = 0.000001) and untreated i.c. tumors (from 30.1% to

14.4%, P = 0.001; Fig. 5A). The frequency of exhausted CD4+ T cells was also reduced in these 2 tumor

locations, albeit to a lesser extent (P < 0.005; Fig. 5A). As reported previously (30), all the TIM3+ T cells were

PD-1+ (Fig. 5A). Like its effect on T-cell exhaustion, Delta-24-RGDOX decreased the frequency of regulatory

T cells in both the virus-injected s.c. tumors (from 20.3% to 5.6%, P = 0.00004) and untreated i.c. tumors (from

30.7% to 14.0%, P = 0.00001) (Fig. 5B). However, exhausted T cells were barely detectable in peripheral blood

and inguinal lymph nodes (Supplementary Fig. S6A), and regulatory T-cell frequency was hardly detected in

blood (Supplementary Fig. S4B, left panel). Although we observed a regulatory T-cell frequency of 89% in

inguinal lymph nodes, the virus treatment had little effect on the frequency of this cell population in the tissue

(Supplementary Fig. S6B, right panel). Interestingly, in all the tissues we examined, we observed the virus-

mediated increase of PD-1+ cytotoxic T cell frequency with the highest in untreated i.c. tumors (Fig. 5C).

Intratumoral injection of Delta-24RGDOX mediates in situ tumor-specific T-cell expansion and

migration to distant untreated tumors

To examine the relevance of the virus-mediated effect on immunity to tumor-specific T-cell response,

we used the OVA protein as a model tumor-associated antigen. We set up the s.c./s.c. tumors as depicted in

Figure 6A. Six days after the implantation of the first tumor and 1 day after the implantation of the second

tumor, OVA-specific CD8+ T cells with luciferase expression (OT-I/Luc T cells) were injected into the first

tumor, followed by 3 doses of viral injections in the same tumor. As demonstrated by bioluminescence imaging

of OT-I/Luc T cells (Fig. 6B), Delta-24-RGDOX treatment promoted the migration of this cell population to

distant OVA+ tumors but not OVA- tumors 4 days after the last dose of viral injection (Fig. 6C). Consistent

with these findings, 3 days later, quantification of OT-I/Luc T cells in the tumors with flow cytometry analysis

revealed that Delta-24-RGDOX treatment increased the number of these cells in the first injected tumor and

greatly enhanced the presence of the cells in the second OVA+ tumors but not OVA- tumors (Fig. 6D),




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suggesting that the virus can activate tumor-specific cytotoxic T cells and increase their in situ presence at the

treated tumor and their migration to distant tumors expressing the T-cell target in this artificial antigen system.

Consistently, Delta-24-RGDOX treatment mediated the increase of OVA-specific CD8+ T cells frequency in

the spleens of the mice bearing s.c. tumors derived from B16-OVA cells (Fig. 6E).

Discussion

Our findings demonstrate that localized treatment with Delta-24-RGDOX induces immunity against

both virus-injected tumors and untreated distant tumors in syngeneic s.c./s.c. and s.c./i.c. melanoma mouse

models. The viral injections into the first s.c. tumor immunologically activate the tumor microenvironment of

not only the treated tumor but also the distant untreated tumors, resulting in the regression of both the treated

primary s.c. tumor and disseminated untreated s.c. and i.c. tumors. In addition, the virus also causes immune

activation in peripheral blood and lymphoid organs. Importantly, the virus treatment stimulates in situ tumor-

specific cytotoxic T-cell expansion and migration to distant untreated tumors expressing the T-cell target.

Based on the evidence from preclinical and clinical experience, unlike ICB, oncolytic virotherapies

should be effective in patients with “cold” tumors. Moreover, localized treatment of the tumor with cancer-

selective oncolytic viruses is expected to cause fewer IRAEs than global immune checkpoint inhibition. In the

phase I clinical trial of Delta-24-RGD in patients with recurrent malignant glioma, patients tolerated intratumal

injection of the virus (up to 3 × 1010

viral particles) well (20). Further, we reported previously that, through

expressing immune stimulator OX40L on cancer cells, Delta-24-RGDOX was more potent than Delta-24-RGD

in inducing tumor-specific immunity and immune memory against gliomas in orthotopic mouse models (21).

During these studies, we observed no virotherapy toxicities, such as weight loss or neurological disorders, in

long-term surviving mice. In our current study of virotherapy with Delta-24-RGDOX in melanoma mouse

models, in the long-term surviving mice, we also found no sign of treatment-related toxicity, such as weight

loss, neurological disorders, or chronic inflammation indicated by swelling and redness at the injection site.

These observations suggest that Delta-24-RGDOX is well-tolerated in tumor-bearing mice.




17

Tumors escape immune surveillance through inactivating tumor-specific T cells in the tissue

microenvironment, posing obstacles in cancer immunotherapy (32). T cells are inactivated through multiple

tumor-intrinsic and -extrinsic immunosuppressive mechanisms (33). One such mechanism is tumor cells’

expression of PD-L1, which ligates to immune checkpoint receptor PD-1 on the surfaces of activated T cells,

natural killer cells, macrophages, and several subsets of dendritic cells, thereby impairing T-cell receptor (TCR)

signaling and CD28 co-stimulation (34-36). Upon TCR activation, PD-1 expression is transiently induced on

naïve T cells, and this expression decreases in the absence of TCR signaling but is maintained by chronic

activation with a persisting epitope target (34). We observed much higher PD-1 expression on CD8+ T cells

from tumors than on T cells from peripheral blood and lymphoid organs (Fig. 5C), which suggests that T cells

have a feedback response to continuous stimulation by tumor antigens within the tumor. As in glioma mouse

models described previously (21), PD-L1 was highly expressed on cultured melanoma cells with or without

Delta-24-RGDOX infection and was greatly upregulated by IFNγ which is a pro-inflammatory cytokine

induced by oncolytic adenovirus in vivo (22) (Supplementary Fig. S7A). Through their ligation to PD-1 on T

cells, these cells stay at an anergy status and allow the immune escape of tumor cells. Delta-24-RGDOX

treatment increased the PD-L1 expression level of melanoma cells from the treated tumors in mice

(Supplementary Fig. S7B) and the upregulation of PD-1 expression on CD8+ T cells from treated and untreated

tumors, peripheral blood, and lymphoid organs (Fig. 5C). Nonetheless, the virus treatment increased the activity

of the immune cells against the tumor cells (Fig. 4E) and mediated the regression of both the treated and

untreated tumors (Fig. 1 and 2), suggesting that PD-1 upregulation alone is not sufficient to inhibit T-cell

activity. Actually, accumulating evidence indicates that PD-1 expression is an early marker of T-cell activation,

allowing the identification of the tumor-reactive CD8+ T-cell fraction in melanoma tumors (37-39). T-cell

inactivation needs the collaboration of PD-1 and other immune checkpoint inhibitors, such as TIM3 and TIGIT

(40, 41). Consistent with the immune activation by the virus, in the present study, Delta-24-RGDOX decreased

the frequency of PD-1+ TIM3+ T cells in treated and untreated tumors despite the increase in PD-1+ T cell

frequency (Fig 5A and C). One study of PD-1targeted therapy in lung cancer patients revealed that the




18

upregulation of PD-1 expression on CD8+ T cells from peripheral blood within 4 weeks of treatment initiation

correlates with clinical benefit (42). Thus, the early Delta-24-RGDOXinduced upregulation of PD-1 on CD8+

T cells from peripheral blood might serve as a biomarker to predict patients' responses to the virotherapy. In

addition, regulatory T cells selectively accumulate in the tumor microenvironment, suppressing T cell immune

responses and activities of antigen-presenting cells, including DCs and macrophages (43). Consistent with the

role of OX40 in inhibiting Foxp3 expression and regulatory T cell induction (44), Delta-24-RGDOX

downregulated the frequency of regulatory T cells in treated and untreated tumors (Fig. 5B), which contributed

to the immune activation within the tumors (Figs. 3, 4C and D).

Melanoma has the potential to metastasize to virtually any anatomical site. Compared with other types

of cancers, melanoma is more likely to metastasize to the brain, and brain metastasis occurs in more than 50%

of patients with advanced disease (45). The median survival time after the detection of brain metastasis is 17–22

weeks (45). The prognosis for patients with melanoma brain metastasis is dismal despite current chemotherapy,

surgery, and radiation options (46). The blood-brain barrier (BBB) limits systemic chemotherapies’ penetration

of the central nervous system (CNS), and the negative side effects of radiotherapy pose challenges to

therapeutic success, and some lesions are not accessible for surgery (46). As with T-VEC virotherapy in

melanoma patients (16), we observed that intratumoral injection of Delta-24-RGDOX into the first s.c. tumor

led to immune activation in distant untreated melanomas (Fig. 4C and D), resulting in the regression of both

treated and untreated i.c. tumors (Fig. 2). Meanwhile, we found T-cell activation in the peripheral blood, spleen,

and inguinal lymph nodes after injection of Delta-24-RGDOX into the first s.c. tumor (Fig. 4A, B, and E).

During brain metastasis, the BBB is selectively disrupted (47). In addition, recent studies indicate that, instead

of being immune-privileged, the CNS actively communicates with the immune system through the lymphatic

vascular system (48, 49). These contribute to the interaction between the CNS and immune system. Thus, the

immune factors induced by localized treatment with Delta-24-RGDOX, including pro-inflammatory cytokines

and immune cells, may reach the i.c. melanoma through BBB leakage or lymphatic circulation. In our study, the




19

anti-tumor immunity mediated by localized virus administration is strong enough to have an abscopal anti-

melanoma effect in the brain (Fig. 2).

Since mouse cells are not optimal host for human adenovirus type 5 replication (50), our current models

have limitations to recapitulate the oncolytic effect of the virus in human melanomas although we detected low

level Delta-24-RGDOX replication in B16F10 Red-FLuc cells (Supplementary Fig. S1D). We expect the virus

will be more potent to replicate in human melanomas to initiate tumor destruction cascade and consequential

anti-melanoma immunity. In addition, although the work has been done in tumor models from B16F10 and its

derivative cell lines, we believe the virus should be able to induce similar effect in other solid tumors. We have

shown previously this virus is efficacious to inhibit gliomas in mouse (21).

In summary, we report for the first time that oncolytic adenovirus Delta-24-RGDOX effectively inhibits

both treated primary s.c. and untreated distant s.c. and i.c. tumors, stimulates systemic immune activation in the

treated mice, and promotes tumor-specific cytotoxic T-cell expansion and their targeting of disseminated

untreated tumors. Our data provide strong evidence that this strategy can be further developed into an

efficacious and safe clinical intervention for patients with advanced melanomas, including those with brain

metastasis.

Authors’ Contributions

Conception and design: H. Jiang, J. Fueyo, C. Gomez-Manzano

Development of methodology: H. Jiang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Jiang

D. H. Shin, T. T. Nguyen, X. Fan, V. Henry, C. Carrillo, Y. Yi, T. L. Collier

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.

Jiang, Y. Yuan

Writing, review, and/or revision of the manuscript: H. Jiang, J. Fueyo, C. Gomez-Manzano, M. M. Alonso




20

Administrative, technical, or material support (i.e., reporting or organizing data, constructing

databases): F. F. Lang

Study supervision: H. Jiang, J. Fueyo, C. Gomez-Manzano

Acknowledgements

We thank Joe Munch (the Department of Scientific Publications, MD Anderson Cancer Center) for editing the

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Figure Legends

Figure 1. Injection of Delta-24-RGDOX into the primary s.c. melanoma inhibits distant untreated s.c.

melanoma and induces immune memory. A, A cartoon depiction of the treatment scheme for the schedule

(left) and position of implantation and viral injection (right). s.c.: subcutaneously; i.t.: intratumorally. B,

Representative bioluminescent images of mice treated with PBS or Delta-24-RGDOX at indicated time points.

C, Spider plots of the tumor bioluminescence in the mice from the indicated treatment groups. D, Survival plots

of the treatment groups (n = 9). E, Survival plots Delta-24-RGDOX treatment survivors after being first re-

challenged with B16F10 Red-FLuc (left panel, n = 4) and then CMT64 (right panel, n = 4) cells. D24-RGDOX:

Delta-24-RGDOX; NS: not significant (P ≥ 0.05); *P < 0.01, log-rank test.

Figure 2. Injection of Delta-24-RGDOX into the s.c. melanoma inhibits distant untreated i.c. melanoma

and induces immune memory. A, A cartoon depiction of the treatment scheme for the schedule (left) and

position of implantation and viral injection (right). s.c.: subcutaneously; i.c.: intracranially; i.t.: intratumorally.

B, Representative bioluminescent images of mice treated with PBS or Delta-24-RGDOX at indicated time

points. C , Spider plots of the tumor bioluminescence in the mice from the indicated treatment groups. D,

Survival plots of the treatment groups. PBS: n = 8; D24-RGDOX: n = 11. E, Survival plots for re-challenging

survivors from Delta-24-RGDOX treatment with B16F10 Red-FLuc-3 (n = 5) cells in the contralateral

hemisphere. F, Shown are representative hematoxylin-and-eosin-stained, whole-mount coronal sections of the

mouse brains from Group Naïve (upper panel, euthanized on Day 26) or Survivor (lower panel, euthanized on

Day 95) from E. Scale bars: 1 mm. D24-RGDOX: Delta-24-RGDOX; NS: not significant (P ≥ 0.05); * P =

0.005, log-rank test.

Figure 3. Effect of Delta-24-RGDOX on tumor cells and lymphocytes at the injected tumor. A, Fourteen

days after s.c. tumor implantation, Delta-24-RGDOX was injected intratumorally. 24 h later, the tumor was

fixed and processed for immunofluorescent staining for E1A. Representative images are shown. Scale bars: 100




26

µm. B, MHC I and MHC II expression in melanoma cells from implanted tumors. After the implantation of

B16F10-EGFP, Delta-24-RGDOX was injected intratumorally on days 7. After 48 hours, the tumors (taken

from 3-4 mice/group) were harvested, dissociated, and analyzed with flow cytometry for MHC I and MHC II

expression. Tumor cells were gated for EGFP+. IgG staining was used as a negative control. The colored

numbers indicate the MFI for the curve of the same color in the graphs. C-F, The mice with 1 s.c. tumor were

treated as depicted in Figure 1A. Three days after the last dose of viral injection, tumors were collected (4-5

mice/group), and the leukocytes in the tumor were enriched and analyzed with flow cytometry to assess the

number of leukocytes (CD45+) (C); the frequency (D) and number (E) of T cells (CD3+), cytotoxic T cells

(CD3+ CD8+) and T helper cells (CD3+ CD4+); and the frequency and number of effector T cells (F). D, Left

panel: Representative flow cytometry plots; the numbers are the frequency of the gated cell populations. Right

panel: quantification of the cell populations in left panel. D24-RGDOX: Delta-24-RGDOX. Teff: effector T

cells. Values represent means ± standard deviations (n = 3). *P = 0.03; **P < 0.0002; NS: not significant (P ≥

0.05); 2-tailed Student t-test.

Figure 4. Systemic immune activation induced by localized Delta-24-RGDOX treatment in tumor-bearing

mice. The mice with s.c. and i.c. tumors were treated as depicted in Figure 2A. Three days after the last dose of

viral injection, tissues were collected from the mice (4-5 mice/group) for immunological analysis. A-D, The

leukocytes from the blood (A and B) and brain hemispheres with tumor implantation (C and D) were analyzed

with flow cytometry to assess the frequency of T cells (CD3+), cytotoxic T cells (CD3+ CD8+), and T helper

cells (CD3+ CD4+) (A and C); and the frequency (B) and number (D) of effector T cells. A and C, Left panel:

Representative flow cytometry plots; the numbers are the frequency of the gated cell populations. Right panel:

quantification of the cell populations in left panel. E, Splenocytes and cells from inguinal lymph nodes (tumor-

draining lymph node of the s.c. tumor) were co-cultured with B16F10 Red-FLuc-3 cells with or without the

infection of the indicated viruses. The amount of IFNγ secreted into the medium was assessed with ELISA forty




27

hours later. D24-RGD: Delta-24-RGD; D24-RGDOX: Delta-24-RGDOX. Teff: effector T cells. Values

represent means ± standard deviations (n = 3). NS: not significant (P ≥ 0.05); *P < 0.05, 2-tailed Student t-test.

Figure 5. Disruption of immune suppression in both treated and untreated tumors by Delta-24-RGDOX.

The mice with s.c. and i.c. tumors were treated as depicted in Figure 2A. Three days after the last dose of viral

injection, the leukocytes from the virus-injected s.c. tumors, peripheral blood, spleens, inguinal lymph nodes,

and brain hemispheres with untreated tumor (4 - 5 mice/group) were analyzed with flow cytometry to assess the

frequency of exhausted T cells (PD-1+ TIM3+) (A) and regulatory T cells (B) and the frequency of PD-1+ cells

in CD8+ T cells (C). A and B, Upper panel: representative flow cytometry plots; the numbers are the frequency

of the gated cell populations. Lower panel: quantification of the cell populations in left panel. Values represent

means ± standard deviations (n = 3). *P < 0.005, 2-tailed Student t-test. D24-RGDOX: Delta-24-RGDOX;

Treg: regulatory T cell.

Figure 6. Tumor-specific T-cell expansion and migration to target distant tumors mediated by localized

Delta-24RGDOX treatment. A, A cartoon depiction of the treatment scheme for the schedule (left) and

position of implantation and viral injection (right). OVA-specific (OT-I/Luc) T cells (1 106) were injected on

day 6. s.c.: subcutaneously; i.t.: intratumorally. B, Bioluminescence imaging shows the distribution of tumor-

specific T cells after i.t. injection of Delta-24-RGDOX in the first implanted s.c. tumor at the indicated times. C,

Quantification of bioluminescence in the second untreated tumors of the mice shown in B on days 7 and 16. D,

On day 19, the lymphocytes from the tumors (5 mice/group) were analyzed with flow cytometry to assess

OVA-Tet+ CD8+ cells. The quantification of this cell population in the tumors is shown. Values represent

means ± standard deviations (n = 3). Groups A, B, and C in C and D are the same as those indicated in B. E,

OVA-specific IFNγ-producing cells detected by ELISPOT. After implantation of B16-OVA cells s.c., three

doses of Delta-24-RGDOX were injected introtumorally on Days 7, 9, 11. On Day 16, splenocytes were isolated

from the mice (3 mice per group) and stimulated with indicated peptides. IFNγ spot-formaing cells (SFC) were




28

measured using an IFNγ ELISPOT assay. D24-RGDOX: Delta-24-RGDOX; NS: not significant (P ≥ 0.05); *P

< 0.05, 2-tailed Student t-test.




Published OnlineFirst August 27, 2019.Clin Cancer Res Hong Jiang, Dong Ho Shin, Teresa T. Nguyen, et al. Disseminated Subcutaneous and Intracranial MelanomasDelta-24-RGDOX Induces Systemic Immunity against Localized Treatment with Oncolytic Adenovirus

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